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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.87, #2)

Semicontinuous production of lipase by Acinetobacter radioresistens in presence of nonwoven fabric by Sheng-Chou Li; Jau-Yann Wu; Chuh-Yung Chen; Teh-Liang Chen (pp. 73-80).

The effect of a hydrophobic nonwoven fabric on the lipase production by Acinetobacter radioresistens was investigated with semicontinuous culture. The fermentation medium contained n-hexadecane as the carbon source. The nonwoven fabric was made from nylon 6 and coated with an acrylic resin. Equipping the nonwoven fabric around the baffles of a 2.5-L agitated fermentor could provide a fine dispersion of n-hexadecane, thus enhancing lipase production. The improvement on lipase yield by using the nonwoven fabric was found to be comparable to that of using an emulsifier (gum arabic). Compared with the corresponding culture in the absence of nonwoven fabric, the employment of the nonwoven fabric could significantly enhance both lipase yield and volumetric productivity.

Keywords: Lipase production; Acinetobacter radioresistens ; semicontinuous culture; nonwoven fabric; dispersion of oil

Production and secretion patterns of cloned glucoamylase in plasmid-harboring and chromosome-integrated recombinant yeasts employing an SUC2 promoter by Hyung Joon Cha; Hee Jeong Chae; Suk Soon Choi; Young Je Yoo (pp. 81-93).

To understand the differences in production and secretion patterns between plasmid-harboring and chromosome-integrated recombinant yeasts, the two recombinant Saccharomyces cerevisiae yeasts, containing the structural glucoamylase STA gene and the SUC2 promoter, were investigated. Both systems were regulated by glucose concentration in the culture broth. First, the glucoamylase activity per gene copy number of the chromosome-integrated recombinant yeast was 2.8- to 5.6-fold higher than that of the plasmid-harboring recombinant yeast. Overburdened owing to high copy number, the plasmid-harboring recombinant yeast gave lower glucoamylase activity per gene copy number. Second, the efficiency of signal sequence was compared; the secretion efficiency of glucoamylase in the plasmid-harboring recombinant yeast was higher than that in the chromosome-integrated recombinant yeast at 96 h of cultivation (74 vs 65%). We postulated that the higher level of secretion efficiency of the plasmid-harboring recombinant yeast resulted because the production level did not reach the capacity of the secretory apparatus of the host yeast. However, the specific secretion rate was much higher in the chromosome-integrated recombinant yeast even though the final secretion efficiency was lower. The lower secretion rate in the plasmid-harboring recombinant yeast could be explained by an adverse effect caused by higher production rate. Finally, the optimal glucose concentration for glucoamylase production in the chromosome-integrated recombinant yeast culture was lower than that in the plasmid-harboring recombinant yeast culture owing to gene dosage effect.

Keywords: Plasmid-harboring recombinant yeast; chromosome-integrated recombinant yeast; SUC2 promoter; glucoamylase; production; secretion

Immobilization of alkaliphilic Bacillus sp. cells for xylanase production using batch and continuous culture by Gashaw Mamo; Amare Gessesse (pp. 95-101).

Agar-immobilized alkaliphilic Bacillus sp. AR-009 cells were used for xylanase production using batch and continuous culture. In a batch culture, maximum enzyme production was observed after 48 h and remained high up to 72 h. In repeated batch cultivation, immobilized cells produced an appreciable level of xylanase activity in seven consecutive batches without any significant decline in productivity. For continuous xylanase production, immobilized cells were packed in a jacketed glass column and sterile medium was continuously pumped. A stable continuous production of xylanase was observed over a period of 1 mo. The volumetric productivity of the continuous culture was 17-fold higher than the batch culture using free cells.

Keywords: Alkaline xylanase; thermostable xylanase; immobilized cell; hemicellulase; Bacillus ; alkaliphile

Effect of Escherichia coli chaperonin GroELS on heterologously expressed human immunodeficiency virus type 1 reverse transcriptase in vivo and in vitro by Gottfried Maier; Elena Manakova; Hermann Heumann (pp. 103-115).

The two subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (HIV-1 RT), p66 and p51, were coexpressed in Escherichia coli along with the E. coli chaperonin system GroEL/GroES. Coexpression increases the yield of heterodimeric HIV-1 RT by a factor of 4 to 5 and improves the nucleic acid binding affinity of HIV-1 RT by a factor of 1.6. We have analyzed the reasons for the improvements. The total increase in yield of HIV-1 RT can be attributed to an accumulation of RT subunits in the cells (factor of about 2.8) and an increased growth of the E. coli cells (factor of about 1.4). One reason for the accumulation in the cells is an improved stability of HIV-1 RT subunits toward bacterial proteases. In vitro studies showed that the nucleic acid binding affinity of HIV-1 RT purified from cells that did not coexpress GroELS was stimulated by adding purified GroELS (approx 1.5-fold), whereas HIV-1 RT stemming from cells coexpressing GroELS was stimulated only marginally (approx 1.1-fold). The in vivo as well as the in vitro studies suggest that the chaperonin interacts with HIV-1 RT and therefore affects the folding process of HIV-1 RT.

Keywords: GroE; chaperonin; human immunodeficiency virus type 1; reverse transcriptase; coexpression

Production of citric acid using immobilized conidia of Aspergillus niger by Emine Bayraktar; Ülkü Mehmetoglu (pp. 117-125).

Conidia of Aspergillus niger were immobilized in calcium alginate gel for the production of citric acid. First, the type of the preactivation medium, together with the preactivation period, was investigated. It was found that A. niger requires a 2-d preactivation period at a 0.05 g/L NH₄NO₃ concentration. Second, preactivated cells were used to determine the effects of nitrogen concentration and the flow rate of oxygen and air on the production of citric acid. Maximum citric acid production was attained with medium containing 0.01 g/L of NH₄NO₃. The rate of citric acid production in the nitrogenous medium was 33% higher when oxygen was used instead of air during the production phase. This corresponds to an increase of 85% when compared to production when neither oxygen nor air was fed into the system. In the nonnitrogenous medium citric acid concentration remained similar regardless of the use of air or oxygen. However, in the nonnitrogenous production medium, citric acid production was not influenced considerably when oxygen was used instead of air. The advantage of using immobilized cells is that production is achieved easily in the continuous system. Therefore, citric acid production was also tested using a packed-bed bioreactor, and an increase in productivity by a factor of 22 was achieved compared to the batch system.

Keywords: Aspergillus niger ; citric acid; immobilization; Ca alginate particles; packed-bed bioreactor

Purification and characterization of an extracellular α-l-arabinofuranosidase from Fusarium oxysporum by Paul Christakopoulos; Petros Katapodis; Dimitris G. Hatzinikolaou; Dimitris Kekos; Basil J. Macris (pp. 127-133).

An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.

Keywords: Fusarium oxysporum ; α-l-arabinofuranosidase; purification; characterization; synergism

Immobilized Streptomyces clavuligerus NP1 cells for biotransformation of penicillin G into deacetoxycephalosporin G by Arnold L. Demain; Marco A. Báez-Vásquez (pp. 135-140).

An investigation was conducted to determine whether immobilized resting cells of Streptomyces clavuligerus NP1, entrapped on a polymeric matrix, are able to perform oxidative ring expansion of benzylpenicillin into deacetoxycephalosporin G by virtue of their deacetoxycephalosporin C synthase (“expandase”) activity. Cells entrapped in polyethyleneimine-barium alginate (1.5%) were able to sustain activity for at least four 2-hcycles, whereas free resting cells were inactive after the second cycle. Although entrapped cells exhibited lower oxidative ring expansion activity than free resting cells, immobilization may offer storage stability, recyclability, and operational stability for biotransformation of penicillins to cephalosporins, thus contributing to the development of a biological means for the production of the important industrial intermediate 7-aminodeacetoxycephalosporanic acid.

Keywords: Penicillins; cephalosporins; biotransformation; immobilized cells; β-lactam antibiotics

Kinetic studies and analytical application of cholesterol oxidase and peroxidase immobilized to synthetic polymer by Lyubov K. Yotova; Ivan P. Ivanov (pp. 141-151).

A method for individual and simultaneous covalent immobilization of cholesterol oxidase and peroxidase to copolymer of acrylonitrile with acrylamide is described. The effect of immobilization on the catalytic properties of the covalently bound enzymes was studied. The immobilized enzymes showed no change in pH optima and an increase in temperature optima, activation energy, and K m, compared to data received from experiments with soluble enzymes. A small glass column packed with immobilized multienzyme complex was used to develop a method for manual determination of cholesterol in foodstuffs (e.g., in mayonnaise “Olinease”). The method was characterized by high analytical precision (coefficient of variation = 2.67%). The results show high correlation with those obtained by the Kageyama method (r=0.986). The method is economical (the enzyme-carrier conjugate may be used more than 300 times), precise, easy to perform, and less time-consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for cholesterol determination in foodstuffs.

Keywords: Immobilization; simultaneous immobilization; cholesterol measurement; cholesterol oxidase; peroxidase; copolymer of acrylonitrile with acrylamide

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.87, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.87, #2)